Method of assaying collagen fragments in body fluids, a test kit and means for carrying out the method and use of the method to diagnose the presence of disorders associated with the metabolism of collagen

ABSTRACT

A method of assaying collagen fragments in body fluids, including bringing a sample of body fluid in contact with at least one immunological binding partner for the collagen fragments, said binding partner being immunoreactive with synthetic peptides, the sequences of which are essentially derived from collagen and containing potential sites for cross-linking. The immunological binding partners are incorporated, either as whole antibodies or as immunologically active fragments thereof, in an assay for quantitative determination of collagen fragments in the sample. In addition to being contacted with the immunological binding partner(s), the sample may be brought into direct contact with the corresponding synthetic peptide. The invention further comprises a test kit and specific means for carrying out the method. The structure of specific peptides is also described.

This is a division of application Ser. No. 08/963,825, filed Nov. 4, 1997, now U.S. Pat. No. 6,110,689, which is a continuation of Ser. No. 08/187,319, filed Jan. 24, 1994, now abandoned.

The present invention relates to a method of determining collagen fragments in body fluids. The invention further relates to means, including synthetic peptides, monoclonal and polyclonal antibodies and cell lines, for use in carrying out the method of the invention. Still further, the invention relates to the use of the above method to diagnose the presence of disorders associated with the metabolism of collagen, especially osteoporosis.

BACKGROUND OF THE INVENTION Collagens and Disorders of Collagen Metabolism

Osteoporosis is the most common bone disease in humans. Primary osteoporosis, accompanied by increased susceptibility to fractures, results from a progressive reduction in skeletal bone mass. It is estimated to affect 15-20 million individuals in the USA alone. Its basis is an age-dependent imbalance in bone remodeling, i.e., in the rates of formation and resorption of bone tissue.

In the USA about 1.2 million osteoporosis-related fractures occur in the elderly each year including about 538,000 compression fractures of the spine, about 227,000 hip fractures and a substantial number of early fractured peripheral bones. Between 12 and 20% of the hip fractures are fatal because they cause severe trauma and bleeding, and half of the surviving patients require nursing home care. Total costs from osteoporosis-related injuries now amount to at least $10 billion annually in the USA (Riggs, New England Journal of Medicine 327:620-627 (1992)).

Osteoporosis is most common in postmenopausal women who, on average, lose 15% of their bone mass in the 10 years after menopause. This disease also occurs in men as they get older and in young amenorrheic women athletes. Despite the major, and growing, social and economic consequences of osteoporosis, the availability of reliable assays for measuring bone resorption rates in patients or in healthy subjects is very limited. Other disorders entailing (and correlated with) abnormalities in collagen metabolism include Paget's disease, Marfan's syndrome, osteogenesis imperfecta neoplastic growth in collagenous tissue, dwarfism, rheumatoid arthritis, osteoarthritis and vasculitis syndrome.

Three known classes of human collagen have been described to date. The Class I collagens, subdivided into types I, II, III, V, and XI, are known to form fibrils. Their full amino-acid sequence (to the extent they have been elucidated) are attached in Appendix A.

Collagen type I accounts for more than 90% of the organic matrix of bone. Therefore, in principle, it is possible to estimate the rate of bone resorption by monitoring the degradation of collagen type I. Likewise, a number of other disease states involving connective tissue can be monitored by determining the degradation of collagen. Examples are collagen type II degradation associated with rheumatoid arthritis and osteoarthritis and collagen type III degradation in vasculitis syndrome.

Amino acid sequences of human type III collagen, human pro 1(II) collagen, and the entire prepro 1(III) chain of human type III collagen and corresponding cDNA clones have been investigated and determined by several groups of researchers; see Loil et al., Nucleic Acids Research 12:9383-9394 (1984); Sangiorgi et al., Nucleic Acids Research 13:2207-2225 (1985); Baldwin et al., Biochem J. 262:521-528 (1989); and Ala-Kokko et al., Biochem. J. 260:509-516 (1989).

Type I, II, and III collagens are all formed in the organism as procollagen molecules, comprising N-terminal and C-terminal propeptide sequences, which are attached to the core collagen molecules. After removal of the propeptides, which occur naturally in vivo during collagen synthesis, the remaining core of the collagen molecules consists largely of a triple-helical domain having terminal telopeptide sequences which are non-triple-helical. These telopeptide sequences have an important function as sites of intermolecular cross-linking of collagen fibrils extracellularly. The alpha-helical region also includes crosslinkable sites. Peptides from this region are part of the present invention.

Intermolecular cross-links provide collagen fibrils with biomechanical stability. The formation of these cross-links is initiated by modification of lysine and hydroxylysine residues to the corresponding aldehydes. Several of these residues located on adjacent chains of collagen will spontaneously form different intermolecular cross-links. The exact position of the sites for cross-linking on collagen telopeptides and from the helical region has been previously described. See, for example, Kühn, K., in Immunochemistry of the Extracellular Matrix 1:1-29, CRC Press, Inc., Boca Raton, Fla. (1982), Eyre, D. R., Ann. Rev. Biochem. 53:717-48 (1984) or U.S. Pat. No. 5,140,103). Furthermore, the amino acid sequences of some potential sites for cross-linking in type I, II, and III collagen are given in Table 1 below.

The fibrous proteins, collagen and elastin, are cross-linked by a unique mechanism based on aldehyde formation from lysine or hydroxylysine side chains. Four homologous loci of cross-linking are evident in molecules of type I, II and III collagens (for review see Kühn, K., in Immunochemistry of the Extracellular Matrix 1:1-29 (1982)). Two are aldehyde sites, one in each telopeptide region. The other two sites are hydroxylysine symmetrically placed at about 90 residues from each end of the molecule. When collagen molecules pack into fibrils, these latter sites in the helical region align and react with telopeptide aldehydes in adjacent molecules. There is now strong evidence that 3-hydroxypyridinium residues are the mature cross-link coming from hydroxylysine-derived aldehydes. The mature cross-linking residues of the other pathway, i.e. from aldehyde formation of lysine residues, is, however, still unknown.

Prior Art Assays for Collagen Degradation

In the past, assays have been developed for monitoring degradation of collagen in vivo by measuring various biochemical markers, some of which have been degradation products of collagen. However, none of these methods are based upon the use of immunological binding partners in the form of antibodies which are immunoreactive with synthetic peptides having a sequence essentially derived from collagen fragments having crosslinkable sites.

For example, hydroxyproline, an amino acid largely restricted to collagen, and the principal structural protein in bone and all other connective tissues, is excreted in urine. Its excretion rate is known to be increased in certain conditions, notably Paget's disease, a metabolic bone disorder in which bone turnover is greatly increased, as discussed further below.

For this reason, urinary hydroxyproline has been used extensively as an amino acid marker for collagen degradation; Singer, F. R. et al., Metabolic Bone Disease, Vol. II (eds. Avioli, L. V., and Kane, S. M.), 489-575 (1978), Academic Press, New York.

U.S. Pat. No. 3,600,132 discloses a process for the determination of hydroxyproline in body fluids such as serum, urine, lumbar fluid and other intercellular fluids in order to monitor deviations in collagen metabolism. The patent states that hydroxyproline correlates with increased collagen anabolism or catabolism associated with pathological conditions such as Paget's disease, Marfan's syndrome, osteogenesis imperfecta, neoplastic growth in collagen tissues and in various forms of dwarfism.

Bone resorption associated with Paget's disease has also been monitored by measuring small peptides containing hydroxyproline, which are excreted in the urine following degradation of bone collagen; Russell et al., Metab. Bone Dis. and Rel. Res. 4 and 5:255-262 (1981), and Singer, F. R., et al., supra.

In the case of Paget's disease, the increased urinary hydroxyproline probably comes largely from bone degradation; hydroxyproline, however, generally cannot be used as a specific index for bone degradation. Much of the hydroxyproline in urine may come from new collagen synthesis (considerable amounts of the newly made protein are degraded and excreted without ever becoming incorporated into tissue fabric), and from turnover of certain blood proteins as well as other proteins that contain hydroxyproline.

Furthermore, about 80% of the free hydroxyproline derived from protein degradation is metabolized in the liver and never appears in the urine. Kiviriko, K. I., Int. Rev. Connect. Tissue Res. 5:93 (1970), and Weiss, P. H. and Klein, L., J. Clin. Invest. 48:1 (1969). Hydroxyproline is a good marker for osteoporosis, but it is troublesome to handle. It is specific for collagen in bones.

Hydroxylysine and its glycoside derivatives, both peculiar to collagenous proteins, have been considered to be more accurate than hydroxyproline as markers of collagen degradation. However, for the same reasons described above for hydroxyproline, hydroxylysine and its glycosides are probably equally non-specific markers of bone resorption; Krane, S. M. and Simon, L. S., Develop. Biochem. 22:185 (1981).

Other researchers have measured the cross-linking compound 3-hydroxypyridinium in urine as an index of collagen degradation in joint diseases. See, for background and as examples, Wu and Eyre, Biochemistry 23:1850 (1984); Black et al., Annals of the Rheumatic Diseases 48:641-644 (1989); Robins et al.; Annals of the Rheumatic Diseases 45:969-973 (1986); and Seibel et al., The Journal of Dermatology 16:964 (1989). In contrast to the present invention, these prior researchers have hydrolyzed peptides from body fluids and then looked for the presence of individual 3-hydroxypyridinium residues.

Assays for determination of the degradation of type I, II, and III collagen are disclosed in U.S. Pat. Nos. 4,973,666 and 5,140,103. However, both these patents are restricted to collagen fragments containing the cross-linker 3-hydroxypyridinium, whereas the present invention does not rely on the presence or absence of this particular cross-linking structure. Furthermore, the above-mentioned assays require tedious and complicated purifications from urine of collagen fragments containing 3-hydroxypyridinium to be used for the production of antibodies and for antigens in the assays.

At present very few clinical data using the approach described in U.S. Pat Nos. 4,973,666 and 5,140,103 are available. Particularly, no data concerning the correlation between the urinary concentration (as determined by methods described in the above-mentioned patents) of 3-hydroxypyridinium containing telopeptides of type I collagen and the actual bone loss (as determined by repeated measurements by bone densitometry) are published. The presence of 3-hydroxypyridinium containing telopeptides in urine requires the proper formation in bone tissue of this specific cross-linking structure at various times before the bone resorbing process. Very little information on these processes is available and the present invention seeks to circumvent this dependance of the correct formation of the cross-linking structure. Furthermore, preliminary data indicate that in one embodiment of the present invention a major fraction of the molecules reactive in the assay has a molecular weight of more than 4,000 daltons. On the contrary, only molecules with a molecular weight below 2,000 daltons are identified in urine with the monoclonal antibody used in the assay; Hanson et al., Journal of Bone and Mineral Research 7:1251-1258 (1992). This demonstrates that the method according to the present Invention has a very different profile of reactivities, i.e. it detects very different molecules, compared to methods described in the two above-mentioned U.S. patents.

None of the above researcher have reported specifically assaying a crosslinkable collagen fragment that is naturally produced in vivo upon collagen degradation, as in the present invention.

GB patent application No. 2,205,643 reports that the degradation of type III collagen in the body can be quantitatively determined by measuring the concentration of an N-terminal telopeptide from type III collagen in a body fluid. This method does not relate to methods employing antibodies reactive with specific, low molecular weight sequences around crosslinkable structures. Instead, the method uses antibodies generated to N-terminal telopeptides released by bacterial collagenase degradation of type III collagen, said telopeptides being labelled and used in the assay.

There are a number of reports indicating that collagen degradation can be measured by quantitating certain procollagen peptides. Propeptides are distinguished from telopeptides and alpha-helical region of the collagen core by their location in the procollagen molecule and the timing of their cleavage in vivo; see U.S. Pat. Nos. 4,504,587; 4,312,853; Pierard et al., Analytical Biochemistry 141:127-136 (1984); Niemela, Clin. Chem. 31/8:1301-1304 (1985); and Rohde et al., European Journal of Clinical Investigation 9:451-459 (1979).

U.S. Pat. No. 4,778,768 relates to a method of determining changes occurring in articular cartilage involving quantifying proteoglycan monomers or antigenic fragments thereof in a synovial fluid sample. This US patent does not relate to detecting collagen fragments derived from degraded collagen.

Dodge, J. Clin, Invest. 83;647-661 (1981) discloses methods for analyzing type II collagen degradation utilizing a polyclonal antiserum that specifically reacts with unwound alpha-chains and cyanogen bromide-derived peptides of human and bovine type II collagens. Contrary to the present invention the degradation products of collagen were not detected in a body fluid, but histochemically by staining of cell cultures, i.e. by “in situ” detection. The main difference between Dodge and the present invention is that Dodge measures type II collagen degradation in situ. By “in situ” is meant a determination carried out in the cells or tissue in which the degradation taken place. There is quite a fundamental difference between this determination and a method based upon tracing a marker in vitro, e.g. in the urine.

None of these references specify the structures of particular telopeptide or alpha-helical degradation products that could be measured to determine the amount of degraded fibrillar collagen in vivo.

SUMMARY OF THE INVENTION

The present invention is based on the discovery of the presence of particular collagen fragments in body fluids of patients and normal human subjects. The collagen fragments are generated upon collagen degradation and are partly characterized by the presence of potential sites for cross-linking, e.g. by the presence of lysine or hydroxylysine (Kühn, K., Immunochemistry of the Extracellular Matrix 1:1-29 (1982)). It is believed that a fraction of the collagen fragments measured by the assays embodied in the present invention are normally covalently linked in vivo to other peptide fragments through different, unidentified or already identified cross-links.

The method of the present invention may be used for determination of the degradation of human collagen of type I, II, and III.

The present invention provides a method of assessing the degradation of collagen based on a determination of the presence and quantity of a particular class of collagen fragments produced in vivo upon collagen degradation; and a comparison of the detected collagen fragments to those of a predetermined standard developed by measuring the same class of collagen fragments in normal individuals, i.e. individuals not afflicted with a disorder affecting collagen metabolism, said individuals being sex- and age-matched with the subjects being tested.

The present invention uses antibodies immunoreactive with synthetic peptides without these cross-linking structures. Accordingly, it is believed that collagen fragments (corresponding to the synthetic peptides) with or without actual cross-links, but with crosslinkable sites, are measured in the assays embodied in the present invention.

In a preferred embodiment, the method is based on the competitive binding of collagen fragments in body fluids and of synthetic peptides essentially derived from collagen to immunological binding partners.

The present invention provides new and very simple procedures for the detection (qualitative and quantitative) of collagen fragments generated upon collagen degradation.

For purposes of the present invention, as disclosed and claimed herein, the following terms are as defined below:

“Antibody”: A monoclonal or polyclonal antibody or immunoreactive fragment thereof (i.e. capable of binding the same antigenic determinant), including—but not limited to—Fab, Fab′, and F(ab′)2 fragments.

“Crosslinkable sites”: loci in collagen telopeptide or helix amino acid sequences containing lysine or hydroxylysine residues which can form cross-links with telopeptides or helical amino acid sequences of other collagen molecules in vivo.

“Crosslinkable peptides”: peptides containing a fragment of the collagen sequence including at least one crosslinkable site.

Test kit: A combination of reagents and instructions for use in conducting an assay.

“Essentially derived” (about structures): Structures with similar antigenicity, i.e. with an ability, above the level of a non-related peptide, to inhibit the binding of any of the mentioned synthetic peptides to an antibody immunoreactive with said synthetic peptide.

It is contemplated that the method may also be used for assaying collagen fragments in animal body fluids, e.g. for determination of the collagen metabolism. It also can be used during clinical testing of new drugs to assess the impact of these drugs on collagen metabolism.

More specifically, the present invention relates to methods for assaying collagen fragments by the use of synthetic peptides corresponding to the above-mentioned sequences of collagen. Generally, these synthetic peptides will have fewer amino acid residues than the entire collagen molecule, often they will have fewer than 10 amino acids. Also, the synthetic peptides, corresponding to molecules present in body fluids, e.g. urine, will have potential sites for cross-linking, preferably lysine or hydroxylysine, incorporated in the structure.

The present invention comprises the determination of collagen fragments by the use of antibodies which are immunoreactive with the above-mentioned synthetic peptides, said peptides each having a sequence derived from collagen fragments having crosslinkable sites.

The invention also includes cell lines (e.g. hybridomas) that produce monoclonal antibodies immunoreactive with the above-mentioned synthetic peptides. The invention further includes monoclonal antibodies produced by the fused cell hybrids, and those antibodies (as well as binding fragments thereof, e.g. Fab) coupled to a detectable marker. Examples of detectable markers include, but are not limited to, enzymes, chromophores, fluorophores, coenzymes, enzyme inhibitors, chemiluminescent materials, paramagnetic metals, spin labels and radioisotopes.

The methods of the invention involve quantitating in a body fluid the concentration of particular collagen fragments derived from collagen degradation. In a representative assay, collagen fragments in the patient's body fluid and a synthetic peptide immobilized on a solid surface are contacted with an immunological binding partner which is immunoreactive with the synthetic peptide. Suitable body fluids are e.g. human urine, blood, serum, plasma and synovial fluid. It is contemplated that the method may also be used e.g. on saliva and sweat. The body fluid may be used as it is, or it may be purified prior to the contacting step. This purification step may be accomplished using a number of standard procedures, including, but not limited to, cartridge adsorption and elution, molecular sieve chromatography, dialysis, ion exchange, alumina chromatography, hydroxyapatite chromatography, and combinations thereof.

The present invention is based on the discovery of simple procedures for quantitation of collagen fragments in body fluids. In a representative procedure, synthetic peptides containing potential sites for cross-linking, are used for the raising of antibodies and subsequently incorporated in the assay for quantitation of collagen fragments generated in vivo by collagen degradation.

The invention also includes test kits useful for quantitating in a body fluid the amount of collagen fragment derived from the degradation of collagen. The kits comprise at least one immunological binding partner, e.g. a monoclonal or polyclonal antibody specific for a peptide derived from the degradation of collagen. If desired, the immunological binding partner of the test kit may be coupled to detectable markers such as the ones described above.

The invention is described in more detail below. Reference is made to the appended drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1, 2 and 3 are typical standard curves for the α1(I)C1 immunoassay (FIG. 1), the a1(I)N1 immunoassay (FIG. 2) and the α2(I)N1 immunoassay (FIG. 3) to be described in more detail in the examples;

FIG. 4 shows the correlation between total pyridinolin (HPLC) and the α1(I)C1 immunoassay,

FIG. 5 shows the correlation between total pyridinolin (HPLC) and the α1(I)N1 immunoassay,

FIG. 6 shows the individual values of the measurement of α1(I)C1 peptide in urine samples from normal, age-matched women, and

FIG. 7 shows the individual values of the measurement of urine samples from women on a hormone replacement therapy and placebo in the α1(I)C1 immunoassay.

DETAILED DESCRIPTION AND BEST MODE OF CARRYING OUT THE INVENTION

In a preferred embodiment of the method according to the invention, the assaying of type I, II and III collagen fragments in urine is performed by an inhibition ELISA (enzyme linked immunosorbent assay) by metering off a sample of urine and contacting the sample with a synthetic peptide having a sequence derived from collagen and with an antibody, which is immunoreactive with the synthetic peptide. The synthetic peptide is immobilized on a solid support. The antibody is raised against the synthetic peptide.

The combined reagents and sample are incubated, and a peroxidase-conjugated (revealing) antibody is added. After another incubation, a peroxidase substrate solution is added. Following short final incubation, the enzyme reaction is stopped, and the absorbance is measured at 450 nm and compared with a standard curve obtained with standard solutions by the same procedure.

Synthetic peptides are used for the preparation of standards. The concentration of synthetic peptide in a stock solution of the relevant synthetic peptide is determined by quantitative amino acid determination. A two-fold dilution of the stock solution is prepared and subsequently used for the construction of the standard curve in the inhibition ELISA.

Preparation of Synthetic Peptides

The preparation of synthetic peptides may be performed according to procedures well known in the art, e.g. by solid-phase peptide synthesis techniques commonly described as “Merrifield synthesis”. Also classical solution phase techniques may be used. Sequences of interest include potential sites for cross-linking (see for example Kühn, K., in Immunochemistry of the Extracellular Matrix 1:1-29 (1982), Eyre, D. R., Ann. Rev. Biochem. 53:717-48 (1984), or U.S. Pat. No. 5,140,103). Examples of such peptides sequences are given in table 1 below.

Regarding the synthetic peptides, it is possible to omit (or add) one or more amino acid residues from (or to) the crosslinkable site sequences without substantial loss of the ability to (a) raise antibodies recognizing the corresponding native collagen fragment or (b) inhibit the bindings of such antibodies to the native fragment. It is possible to use longer collagen fragments and/or chimeric peptides to raise the antibodies and, in principle, it is not necessary to use the same peptide as the immunogen and the competitor in the assay.

Collagen type I Potential sites in telopeptides: N                        C α1 (I) N-term. Asp-Glu-Lys-Ser-Thr-Gly-Gly (α1(I)N1) (SEQ. ID NO: 1) α1 (I) C-term. Glu-Lys-Ala-His-Asp-Gly-Gly-Arg (α1(I)C1) (SEQ. ID NO: 2) α2 (I) N-term. Gln-Tyr-Asp-Gly-Lys-Gly-Val-Gly (α2(I)N1) (SEQ. ID NO: 3) α2 (I) C-term. no potential sites Potential sites in helix: α1 (I) (near N) Gly-Met-Lys-Gly-His-Arg (SEQ. ID NO: 4) α1 (I) (near C) Gly-Ile-Lys-Gly-His-Arg (SEQ. ID NO: 5) α2 (I) (near N) Gly-Phe-Lys-Gly-Ile-Arg (SEQ. ID NO: 6) α2 (I) (near C) Gly-Leu-Pro-Gly-Leu-Lys-Gly-His-Asn (SEQ. ID NO: 7) Potential sites in telopeptides: N                         C α1 (II) N-term. Pro-Gly-Pro-Lys-Gly-Glu (SEQ. ID NO: 8) Gly-Gln-Lys-Gly-Glu-Pro (SEQ. ID NO: 9) Gly-Asp-Ile-Lys-Asp-Ile-Val (SEQ. ID NO: 10) α1 (II) C-term. Glu-Lys-Gly-Pro-Asp (SEQ. ID NO: 11) Potential sites in helix: αl (II) (near N) Gly-Val-Lys (SEQ. ID NO: 12) Pro-Gly-Val-Lys-Gly (SEQ. ID NO: 13) Potential sites in telopeptides:  N                        C αl (III) N-term. Asp-Val-Lys-Ser-Gly-Val (SEQ. ID NO: 14) αl (III) C-term. Glu-Lys-Ala-Gly-Gly-Phe-Ala (SEQ. ID NO: 15) in helix: αl (III) (near N) Gly-Phe-Pro-Gly-Met-Lys-Gly-His-Arg (SEQ. ID NO: 16) αl (III) (near C) Gly-Ala-Ala-Gly-Ile-Lys-Gly-His-Arg (SEQ. ID NO: 17)

Preparation of Antibodies

The methods for preparation of both monoclonal and polyclonal antibodies are well known in the art. For example, see Campbell, A. M., Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 13 (1986). It is possible to produce antibodies to synthetic peptides by immunization. However, because of the relatively small molecular weight of these compounds it is preferred that the hapten be conjugated to a carrier molecule. Suitable carrier molecules include, but are not limited to, bovine serum albumin, thyroglobulin, ovalbumin, tetanus toxoid, and keyhole limpet hemocyanin. The preferred carrier is bovine serum albumin. To present the hapten in its most immunogenic form to the antibody producing cells of the immunized animal a number of alternative coupling protocols can be used. Suitable procedures include, but are not limited to, glutaraldehyde, carbodiimide, and periodate. Preferred binding agents are glutaraldehyde and carbodiimide.

The preparation of antibodies is carried out by conventional techniques including immunization with collagen fragments or synthetic peptides conjugated to a carrier. To improve the immunogenicity it is preferred that the immunogen be mixed with an adjuvant before injection. Examples of adjuvants include, but are not limited to, aluminum hydroxide, Freund's adjuvant, and immune-stimulating complexes (ISCOMs). ISCOMs can be made according to the method described by Morein, B. et al., Nature 308:457-460 (1984).

Either monoclonal or polyclonal antibodies to the hapten carrier molecule can be produced. For the production of monoclonal antibodies it is preferred that mice are immunized. Spleen cells from the immunized mouse are harvested, homogenized, and thereafter fused with cancer cells in the presence of polyethylene glycol to produce a cell hybrid which produces monoclonal antibodies specific for peptide fragments derived from collagen. Suitable cancer cells include, but are not limited to, myeloma, hepatoma, carcinoma, and sarcoma cells. Detailed descriptions of the production of monoclonal antibodies are provided in Goding, J. W., in Monoclonal Antibodies: Principles and Practice, (1986). A preferred preliminary screening protocol comprises the use of synthetic peptides conjugated to a carrier and coated onto the solid surface of a microtitre plate.

For the preparation of polyclonal antibodies, which are reactive with peptide fragments derived from collagen, different animal species can be immunized. Suitable species include, but are not limited to, chicken, rabbit and goat. Chicken and rabbit are preferred.

Antibody fragments are prepared by methods known in the art (see E. Ishikawa, J. of Immunoassay 3:209-327, 1983).

Conduct of Immunoassays

Accordingly, by utilization of an immunoassay with the antibodies prepared as above it is possible to assay a biological fluid sample without prior fractionation or hydrolysis. The specificity for the desired collagen in the biological fluid is supplied by the antibody in combination with the use of a synthetic peptide (against which the antibody was raised or in any event with which the antibody is immunochemically reactive) in the assay construction. The immunoassays themselves are conducted using any procedure selected from the variety of standard assay protocols generally known in the art. As it is generally understood, the assay is constructed so as to rely on the interaction between the specific immunological binding partner and the desired analyte for specificity and to utilize some means to detect the complex formed by the analyte and the immunological binding partner. The immunological binding partner may be complexed to a solid support and used as a capture immunological binding partner for the analyte. This protocol may be run in a direct form, wherein the formation of analyte/immunological binding partner complex is detected, e.g. by a fluorescent, radioactive or enzymatic label, or it may be run in a competitive format wherein a labelled standard competes with the analyte for the immunological binding partner. The format may also be constructed as an agglutination assay or the complex may be precipitated by addition of a suitable precipitant to the reaction mixture. The specific design of the immunoassay protocol is open to a wide variety of choice, and the number of clinical assay devices and protocols available in the art is multitudinous. For a variety of such protocols, see U.S. Pat. No. 5,001,225.

The antibodies and revealing reagents for the conduct of an immunoassay using standard detection protocols, for example radioisotope labelling, fluorescent labelling or ELISA, either in a direct or competitive format, may conveniently be supplied as kits which include the necessary components and instructions for the assay. In one embodiment of the invention such a kit includes a microtiter plate coated with a relevant synthetic peptide, standard solutions for preparation of standard curve, a urine control for quality testing of the analytical run, rabbit antibodies reactive with the above-mentioned synthetic peptide, anti-rabbit immunoglobulins conjugated to peroxidase, a substrate solution, a stopping solution, a washing buffer and an instruction manual.

Since immunoassays can be constructed using antibodies and specific synthetic peptides, the ratios of the corresponding collagen fragment sequences in an appropriate biological fluid can be determined as well as their individual levels and their total. Thus, the assay can be designed to include antibodies which will result in determination of several native peptide sequences or determination of a single peptide sequence, or any desired combination thereof.

In addition to the use of the herein specified peptides as indicators of bone resorption, bone metabolic balance is advantageously determined by the substantially simultaneous determination of a marker of the formation of bone in the same or other appropriate biological fluid from the same individual. “Substantially simultaneous” means the same day, preferably within 4 hours. For example such markers include osteocalcin (also known as bone GLA protein of BGP), procollagen type I, bone alkaline phosphatase and total alkaline phosphatase. Suitable methods for the determination of these markers can be found, for example, in Delmas, P. D., et al., J. Bone Min. Res. 1:333-337 (1986).

The assay of the present invention which provides an index to determination of the metabolic status of tissues, which generate collagen-derived peptides when degradation occurs, are useful {circumflex over ( )} in a variety of contexts. First, when considering the degradation of type I collagen, the assays are methods to assess an abnormal condition of a subject by indicating, for example, excessive bone resorption. This may show the presence of an osteoporotic condition or the metastatic progress of a malignancy. Other conditions characterized by excessive bone resorption include Paget's disease and hyperparathyroidism. Likewise, a number of other disease states involving connective tissue may be monitored by determination of the degradation of collagen. Examples are collagen type II degradation associated with rheumatoid arthritis and osteoarthritis and collagen type III degradation in vasculitis syndrome. Since the condition of the subject can be monitored continuously, application of these assays can also be used to monitor the progress of therapy administered to treat these or other conditions. Further, the assays can be used as a measure of toxicity, since the administration of toxic substances often results in tissue degradation.

Thus the assays may be applied in any situation wherein the metabolic condition of collagen tissues can be used as an index of the condition, treatment, or effect of substances directly administered to the subject or to which the subject is exposed in the environment.

The following examples are intended to illustrate, but not to limit the invention.

EXAMPLE 1 Immunoassays for Specific Peptide Sequences in Urine

Three peptides (α1(I)C1, α1(I)N1, and α2(I)N1) (see Table 1, p. 13) prepared by solid-phase techniques are used for the preparation of immunogens. For immunization, the peptides are covalently attached to bovine serum albumin using glutaraldehyde reagents and methods well known in the art. Both monoclonal and polyclonal antibodies are raised against the peptides. For production of monoclonal antibodies, Balb/c mice are immunized with peptide-BSA conjugates, and hybridoma cell lines are prepared using standard techniques after fusion of cells from the spleen or lymph nodes with Ag8 myeloma cells. Polyclonal antibodies are raised in rabbits and chicken. Screening of both antisera and hybridoma cell media were performed by ELISA using microtiter plates coated with the appropriate peptide-gelatin conjugate prepared using carbodiimide reagents and methods well known in the art.

Assays for three of the peptide sequences (α1(I)C1 α1(I)N1, and α2(I)N1) in urine are performed by an inhibition ELISA as follows:

Urine samples (10 or 25 1) possibly containing collagen fragments or solutions containing 0.05-15 g peptide/ml as reference standards, respectively, are added to 75 1 of immunological binding partners for the peptides diluted 1:5,000-1:20,000 in phosphate buffered saline containing 0.1% Tween-20 detergent (PBS-T) and including 0.1% (w/v) of BSA. Each sample is prepared in duplicate in flat-bottomed, 96-well microtiter plates previously coated with gelatine conjugate containing the appropriate peptide. After 60 minutes, the plates are washed with PBS-T (3 times) and the bound antibodies are detected by standard techniques with a horse radish peroxidase labelled antibody prepared against the species of the primary antibody. Peroxidase substrate is added and the color development is measured at 450 nm in an automated microtiter plate reader after stopping the enzyme reaction using 1 M H3PO4. Samples containing the analyte decrease the binding of primary antibody to the immunobilized peptide in the plate and thus have a reduced color concentration. The amount of analyte in the sample is quantified with reference to previously established curves from standards included on each plate computed using log-lin plots. FIGS. 1, 2 and 3 show typical standard curves for the α1(I)C1 immunoassay (FIG. 1), α1(I)N1 immunoassay (FIG. 2), and α2(I)N1 immunoassay (FIG. 3).

EXAMPLE 2 Correlation to Pyridinolin Determination on HPLC

On a number of unselected urine samples the concentration of total pyridinolin (HPLC method, see for example Uebelhart, D., Bone and Mineral 8:87-96 (1990)) was measured. Values obtained in this HPLC system were correlated to the values obtained in two immunoassays (α1(I)C1 peptide and α1(I)N1 peptide).

FIG. 4 shows the correlation between total pyridinolin (HPLC) and α1(I)C1 immunoassay (n=59). The correlation calculated by linear regression analysis is r=0.80.

FIG. 5 shows the correlation between total pyridinolin (HPLC) and α1(I)N1 immunoassay (n=36). The correlation calculated by linear regression analysis is r=0.95.

EXAMPLE 3 Clinical Results

The immunoassay procedure (using the α1(I)C1 peptide) set forth in Example 1 was applied to urine samples from different individuals, and the amount of analyte was quantitated. The values obtained were related to the level of urinary creatinine in the urine sample as is commonly done for urine assays. The values obtained for normal, age-matched women (premenopausal and postmenopausal) are shown in Table 2.

TABLE 2 Measurement of α1(I)C1 peptide in urine samples from normal, age-matched women (premenopausal and postmenopausal) Group α1(I)C1 peptide (g/mol creatinine) Premenopausal (n = 104) 0.263 ± 0.143 Postmenopausal (n = 180) 0.426 ± 0.190

For individual values see FIG. 6.

The difference between the premenopausal and the postmenopausal values is highly significant (P<0.0001).

The z-score of a quantitative test procedure shows the ability of the procedure to distinguish between two populations. Table 3 below shows the z-score of the IC1 immunoassay and the state of the art measurement of pyridinolin on HPLC when applied to the same set of urine samples from normal, age-matched premenopausal (n=104) and postmenopausal women (n=180).

TABLE 3 Z-scores obtained for two different test procedures (total pyridinolin (HPLC) and α1(I)C1 immunoassay) when applied to urine samples from normal, age-matched women (premenopausal and postmenopausal) Test procedure Z-score α1(I)C1 immunoassay 1.14 Total pyridinolin (HPLC) 1.25

As can be seen from Table 3, the abilities of the two methods to distinguish between patient populations are approximately alike.

For an assay to be used as an index of bone resorption it is very important to be able to measure the impact of a hormone replacement therapy (HRT). Table 4 shows the results from the α1(I)C1 immunoassay in such a study.

TABLE 4 Measurement of urine samples from women on a hormone replacement therapy and placebo in the α1(I)C1 immunoassay. Values given as percent of t = 0 α1(I)C1 peptide (μg/mol creatinine) Group t = 0 t = 12 months HRT (n = 92) 0.457 ± 0.176 0.176 ± 0.103 Placebo (n = 45) 0.459 ± 0.209 0.402 ± 0.187

For individual values see FIG. 7.

A highly significant drop in the group receiving HRT is seen after 12 months (P<0.001).

All cited patents, patent applications and literature are incorporated by reference in their entirety. In case of conflict, however, the present disclosure controls.

The invention has been described above by reference to specific embodiments. It will be apparent to those skilled in the art, however, that many additions, deletions and modifications are possible without departing from the spirit of the invention as claimed below. 

What is claimed is:
 1. A method of assaying Type I collagen fragments in a body fluid, comprising: (a) contacting a sample of the body fluid with a synthetic peptide immobilized on a solid support and with an immunological binding partner which is immunologically reactive with said immobilized synthetic peptide, wherein the collagen fragments compete with said synthetic peptide for binding with the immunological binding partner; and (b) quantifying the amount of collagen fragments in the body fluid by measuring the amount of binding of said immunological binding partner with said synthetic peptide; wherein said immunological binding partner is immunologically reactive with a peptide immobilized on a solid support and consisting essentially of the amino acid sequence Gln-Tyr-Asp-Gly-Lys-Gly-Val-Gly (SEQ. ID NO: 3); and said immobilized synthetic peptide contains no collagen cross-link structure but spans a region of a sequence of Type I collagen at which in nature a cross-link structure would form, and is immunologically reactive with said immunological binding partner.
 2. The method of claim 1, wherein said immobilized synthetic peptide consists of fewer than 10 amino acids.
 3. The method of claim 1 or 2, wherein the immobilized synthetic peptide consists essentially of the amino acid sequence Gln-Tyr-Asp-Gly-Lys-Gly-Val-Gly (SEQ. ID NO: 3) or consists essentially of the sequence Gln-Tyr-Asp-Gly-Lys-Gly-Val-Gly (SEQ. ID NO: 3) modified by the addition or omission of one or more amino acids while retaining the ability to raise antibodies recognising a corresponding native collagen fragment or to inhibit the binding of such antibodies to a corresponding native collagen fragment.
 4. The method of claim 1, wherein said immunological binding partner is a whole antibody.
 5. The method of claim 4, wherein said antibody is a monoclonal antibody.
 6. A test kit for assaying Type I collagen fragments in a body fluid, comprising: (a) an immunological binding partner which is immunologically reactive with a synthetic peptide immobilized on a solid support and consisting essentially of the amino acid sequence Gln-Tyr-Asp-Gly-Lys-Gly-Val-Gly (SEQ. ID NO: 3); and (b) a synthetic peptide immobilized on a solid support, said synthetic peptide containing no collagen cross-link structure but spanning a region of a sequence of Type I collagen at which in nature a cross-link structure would form, and being immunologically reactive with said immunological binding partner.
 7. The kit of claim 6, wherein said immobilized synthetic peptide consists of fewer than 10 amino acids.
 8. The kit of claim 6 or 7, wherein the immobilized synthetic peptide consists essentially of the sequence Gln-Tyr-Asp-Gly-Lys-Gly-Val-Gly (SEQ. ID NO: 3) or consists essentially of the sequence Gln-Tyr-Asp-Gly-Lys-Gly-Val-Gly (SEQ. ID NO: 3) modified by the addition or omission of one or more amino acids while retaining the ability to raise antibodies recognising a corresponding native collagen fragment or to inhibit the binding of such antibodies to a corresponding native collagen fragment.
 9. The kit of claim 6, wherein said immunological binding partner is a whole antibody.
 10. The kit of claim 9, wherein said antibody is a monoclonal antibody.
 11. A method of assaying collagen fragments in a body fluid which comprises contacting an immunological binding partner with the body fluid and a synthetic peptide, wherein the immunological binding partner is immunoreactive with the immobilized synthetic peptide and with an immobilized peptide consisting essentially of the amino acid sequence Gln-Tyr-Asp-Gly-Lys-Gly-Val-Gly (SEQ. ID NO: 3), and wherein the synthetic peptide contains no collagen cross-link structures and is immobilized on a solid surface. 